A number of gene transfer methods have been developed so far, which include those using viral vectors such as retrovirus or adenovirus, liposomal vectors composed of cationic amphiphilic lipids, DEAE dextran method, gene transfer methods using synthetic polymers or even gene gun.
Viral vectors usually show high gene transfer efficiency, whereas, they have many demerits: It is rather hard to maintain gene expression for desired periods after gene transfer by using viral vectors. Viral vectors may stimulate host immune defense mechanism, induce inflammation and thus may even aggravate disease status especially on repeated administration. It is hard to selectively transfer viral vectors to target cells or target tissues. It takes rather a long time to cultivate and to prepare enough amount of viral vectors. In addition, viral vectors can induce cancer by integration of viral DNA into host chromosomes and may even provoke fatal events upon genetic recombination with latent homologous virus.
Because of the problems associated with viral vectors, non-viral vectors have recently emerged as safe and promising gene transfer tools, which include cationic lipids, synthetic or natural polymers or particles. These non-viral vectors commonly have limited efficiency of transfection (ie. gene transfer). However, recent remarkable advance of synthetic vectors have made them highly promising tools for gene transfer.
Highly valuable information on gene transfer using cationic liposome are listed in the following representative literatures: Hazinski et al, Science, 249, 1285-1288, 1991; Wang and Huang, Proc. Natl. Acad. Sci. (USA), 84, 7851-7855, 1987; Felgner, et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7416, 1987. As compared with viral vectors, liposomal gene vectors have important merits: They can be safely administered into the body via a number of routes, which include direct injection into target tissue, administration via skin, gastrointestinal tract, or airway and they also can be administered systemically by intravenous injection. Nowadays new synthetic vectors are being systematically designed based on their molecular structure and these are entirely different from existing non-viral gene transfer tools which include calcium phosphate precipitaion, or cationic polymer such as DEAE dextran, polylysine and polybrene, or physical gene transfer methodology such as gene gun or electroporation. Novel synthetic vectors hold reasonable promises of highly valuable medicine which are essential for successful gene therapy and are expected to have big market in the future.
The conventional methods of gene therapy included ex vivo gene transfer in which gene was initially transferred into cells in vitro and then these cells transferred by gene were administered into the body. However, this type of ex vivo gene therapy was too much complicated in methodology and costs too much money to be commonly applied in clinical practice. Nowadays the focus of gene therapy is moving from ex vivo gene therapy to in vivo gene therapy. The goal of modern gene therapy is to develop systemic gene therapy method which can transfer gene efficiently and specifically to target cells without significant toxicity. In this regard, some nonviral vectors are focus of interest and research, which include lipid carrier, in particular cationic liposome, molecular conjugates which transfer genes depending on receptor-mediated endocytosis, and synthetic biopolymer vectors.
Cationic lipid molecules make complex particles with negative charged-DNA molecules by forming stable ionic bonding and these liposome-DNA complex enter inside cells by forming fusion with cell membrane or by natural endocytosis process. Felgner first reported on the use of cationic lipids for gene transfer on 1987 (Felgner, et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7416, 1987). He showed that cationic liposome made of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium chloride (DOTMA), an amino lipid, and dioleoylphosphatidyl ethanolamine (DOPE), a lipid with cell membrane fusion-activity was useful for gene transfer, since when a variety of cationic lipid molecules have been developed and used for gene transfer. DOTMA, a cationic lipid with high gene transfer efficiency, has hydrophobic radical which are composed of C18-aliphatic group with double bonds. With regard to structure of DOTMA, the fourth ammonium salts are bound to the opposite site of hydrophobic radicals which are bound by 3-carbon spacer arm and double ether linker bond. DOTMA has high gene transfer efficiency, but carries high cellular toxicity and also requires a number of lengthy synthetic steps. The mixture of DOTMA and DOPE is being commercially sold in the brand name of Lipofectin.
Cationic lipid molecules used for gene transfer are amphipathic compounds and commonly are composed of 3 parts, including cationic head, spacer and hydrophobic tail. Hydrophobic tail part is usually made of fatty acid derivatives such as oleic acid or myristic acid. Cationic ammonium radical acts as an anchor for the contact of the surface of liposome and cell membrane. Glycerol is included as spacer. From 2 to 15 of carbons are hydrophilic. The first, second, third or fourth ammonium radicals with positive charge at neutral pH are included as cationic head.
Novel derivatives of DOTMA have been developed to increase gene transfer efficiency, which include 1,2-dimyrisyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) and 2,3-dioleyloxy-N-[2-(sperminecarboxyamide)ethyl]-N,N-dimethyl-1-propane ammonium trifluoroacetate (DOSPA).
In addition, some cationic lipid derivatives of cholesterol have been developed for gene transfer, which include 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol), dimethyl-dioctadecyl ammonium bromide (DDAB), N-(α-trimethylammonio acetyl)-didodecyl-D-glutamate chloride (TMAC) and dioctadecylamidoglycyl-spermine (DOGS). Depending on their structures and number of positive charges, these cationic lipids are classified into 1) those which provide one positive charge by combination of head of lipid and the fourth amine, third amine, or hydroxyethylic fourth amine and 2) those which provide multiple positive charges by combination of lipids with spermine or polylysine.
Another type of cationic lipid used for gene transfer are detergents composed of the fourth ammonium salt, which include single chain detergent such as cetrimethylammonium bromide and double chain detergent such as dimethyldioctadecyl ammonium bromide. These detergents can transfer gene into animal cells. Amino radicals of these amphiphilic detergents are the fourth radical and single chain is united with the first amine radical without spacer arm or linker bond. These amphiphiles show cellular toxicity on administration to mammalian cells.
Another type of amphiphilic molecules tested include 1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol (DOBT), cholesteryl(4′-trimethyl-ammonino)butanoate (choTB) and 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) all of which have similar structure to that of DOTMA and but have limited gene transfer efficiency.
Two types of L-5-carboxyspermine, amphiphilic molecules with the first and second ammonium radicals, have been developed and used for gene transfer, which are often called as lipopolyamine and include dioctadecyl-amidologlycylspermine (DOGS) and dipalmitoyl phosphatidyl ethanolamido-spermine (DPPES). These two amphiphilic molecules are particularly useful to transfect primary endocrine cells without significant cellular toxicity.
Lipopolylysine was also reported to be a mediator of gene transfer, the structure of which contains phospholipid(N-glutaryl-phosphatidyl ethanolamine) and polylysine as ammonium radical. Side chain of lysine and head group of phospholipid are spacer arm and hydrophobic group is a double chain united with spacer arm by double ester bonds. Lipopolysine shows high gene transfer efficiency and is devoid of significant cellular toxicity, but shows gene transfer activity only on scraping treatment of target cells, and therefore is inconvenient and of little value in in vivo gene transfer.
Liposome is a microscopic vesicle which has lipid bilayer. The shape and structure of liposome are highly variable from long tube structure to globular shape. The diameter of liposome range from hundreds of Å to several millimeter. The lipid bilayer structure of liposome is composed of hydrophilic layer and concentrated lamellas. The diameter of liposomal vesicle usually range from 20 to 30,000 nm and liquid film layers which are located between lamellas 3 to 10 nm, respectively. Liposome is classified into 3 types depending on their overall size and characteristics of lamellar structure: multilamellar vesicle (MLV), small unilamellar vesicle (SUV) and large unilamellar vesicle (LUV). SUV generally have diameter of from 20 to 50 nm and their structure are composed of single lipid bilayer surrounding central hydrophilic area. Unilamellar vesicle is produced in diameter of from 50 to 600 nm. Unilamellar vesicle is a single compartment vesicle with uniform size, whereas, MLV has a highly variable size with diameter up to 10,000 nm and multicompartmental structure and contains one or more bilayers. Large LUV has diameter of from 600 to 30,000 nm and contain more than 1 bilayer.
The methods of synthesis of liposome are manifold, but generally three types of methods are used, which are as follows. The first is ultrasonic dispersion method in which metal probe is immersed into the suspension of MLV. This method is commonly used to produce SUV. The second is synthetic method of MLV liposome in which lipids are dissolved in appropriate organic solvent and solvent is removed by gas or air, and then remaining thin membrane of dried lipids are mixed with solution and shaked, and finally lipids are dispersed in the form of lipid aggregates or liposome. The third is synthetic method of LUV liposome in which thin membrane of lipids are slowly hydrated by using distilled water or several types of solution. The other method is based on freezing lyophilization by which lipid film is produced. Lipid film is dissolved in volatile solvent, frozen and then solvent is removed again by using lyophilizer. Addition of drug solution to lyophilized lipid produce liposome which is used as pharmaceutical formulation carrying drugs into the body. Various methods of synthesis of liposome are reported in the literatures and patents (Felgner, P. L. and Ringold, G. M., Nature, 337, 387˜388, (1989); WO93/05162, U.S. Pat. Nos. 5,994,317; 6,056,938).
As described above, liposome are basically synthesized from one or more lipid molecules. A variety of lipids including cationic lipids, neutral lipids or anionic lipids are used to synthesize liposome. In particular cationic lipids have most widely been used to synthesize liposome for gene transfer, but they have several problems: Amines which have been used to make many cationic liposome are chemically unstable and have only short shelf life of vesicle. For example, dimethyl dioctadecyl ammonium bromide, a type of amine, lacks appropriate molecular bonding which are necessary for formation of appropriate lipid bilayer structure of liposome.
To manufacture pharmaceutical preparations of liposome, so called encapsulation of biologically active materials by liposome are necessary, which is carried out by mixing of the materials with lipid followed by formation of liposome. The problems common to the above encapsulation process are that less than 50%, usually only less than 20% of biologically active target materials are encapsulated by liposome, thus another process is necessary to remove un-encapsulated materials, and this removal process may induce damage of encapsulating liposome. Therefore, maintenance of stability of liposome is critical in encapsulation process.
Liposome are most commonly used non-viral DNA transfer vehicle, but other methods also have been in use for gene transfer, which include microinjection, protoplast fusion, liposome fusion, calcium phosphate precipitation, electroporation and retrovirus, etc. Each of the methods has its own merits and demerits. In particular, these methods have only limited gene transfer efficiency, but induce significant toxicity and are so complicated in methodology, which precluded their use in large scale clinical trial of biological therapy or gene therapy. For example, calcium phosphate precipitation method has gene transfer efficiency of only 1 cell out of 104 to 107 cells. Microinjection is efficient in gene transfer to a limited number of cells in vitro, but it is impossible to apply to large number of cells or patients. Protoplast fusion have higher gene transfer efficiency than that of calcium phosphate precipitation method, but requires propylene glycol, which induces significant cellular toxicity. Electroporation method has high gene transfer efficiency but requires special apparatus. Retrovirus has pretty good gene transfer efficiency, but may induce viral infection or cancer to the host which precludes their widespread application in human patients. Liposome have been commonly used for gene transfer, but most of the old style liposome have only limited gene transfer efficiency not so much higher than that of calcium phosphate precipitation method. The ideal method of gene transfer must have high gene transfer efficiency, no cellular toxicity and be free from contamination by infective material and should be simple in methodology so that it does not require complicated apparatus or expensive machine.